Neurofibromin Deficiency and Extracellular Matrix Cooperate to Increase Transforming Potential through FAK-Dependent Signaling.

Andrea Errico,Franco Bassetto,Alberto Gambalunga,Gianluca Tadini,Anna Stocco,Federica Chiara,Stefano Ferraresi,Andrea Trevisan,Martina Grigatti,Amedeo Ferlosio,Vincent M Riccardi,Andrea Rasola,Sandra Giustini,Debora Garozzo

doi:10.3390/cancers13102329

Abstract

Plexiform neurofibromas (Pnfs) are benign peripheral nerve sheath tumors that are major features of the human genetic syndrome, neurofibromatosis type 1 (NF1). Pnfs are derived from Schwann cells (SCs) undergoing loss of heterozygosity (LOH) at the NF1 locus in an NF1+/- milieu and thus are variably lacking in the key Ras-controlling protein, neurofibromin (Nfn). As these SCs are embedded in a dense desmoplastic milieu of stromal cells and abnormal extracellular matrix (ECM), cell-cell cooperativity (CCC) and the molecular microenvironment play essential roles in Pnf progression towards a malignant peripheral nerve sheath tumor (MPNST). The complexity of Pnf biology makes treatment challenging. The only approved drug, the MEK inhibitor Selumetinib, displays a variable and partial therapeutic response. Here, we explored ECM contributions to the growth of cells lacking Nfn. In a 3D in vitro culture, NF1 loss sensitizes cells to signals from a Pnf-mimicking ECM through focal adhesion kinase (FAK) hyperactivation. This hyperactivation correlated with phosphorylation of the downstream effectors, Src, ERK, and AKT, and with colony formation. Expression of the GAP-related domain of Nfn only partially decreased activation of this signaling pathway and only slowed down 3D colony growth of cells lacking Nfn. However, combinatorial treatment with both the FAK inhibitor Defactinib (VS-6063) and Selumetinib (AZD6244) fully suppressed colony growth. These observations pave the way for a new combined therapeutic strategy simultaneously interfering with both intracellular signals and the interplay between the various tumor cells and the ECM.

Highlights

Neurofibromatosis type 1 (NF1), known as von Recklinghausen disease, is an autosomal dominant disease caused by inactivating mutations of the neurofibromatosis type 1 (NF1) gene coding a 2818 amino acid protein, neurofibromin (Nfn).The trademark of NF1 is the development of multiple benign peripheral nerve sheath tumors called neurofibromas
Mouse embryonic fibroblasts (MEFs) cells isolated from wild type and Nf1−/− mice [37] as a preliminary experimental system (Nf1+/+ and Nf1−/− MEFs; Figure 1a)
PDGF-BB stimulation (Figure 1d) and further potentiated when we added a mixture of Collagen/Fibronectin to the Matrigel matrix (Figure 1e). These results suggest a cooperative contribution of pathways elicited by Nf1 loss and by focal adhesion kinase (FAK) activation in fostering neoplastic growth

Summary

Introduction

Neurofibromatosis type 1 (NF1), known as von Recklinghausen disease, is an autosomal dominant disease caused by inactivating mutations of the NF1 gene coding a 2818 amino acid protein, neurofibromin (Nfn).The trademark of NF1 is the development of multiple benign peripheral nerve sheath tumors called neurofibromas. Neurofibromas are complex tumors originating from the peripheral nerve sheath, composed of Schwann cells (SCs) undergoing loss of heterozygosity (LOH) at the NF1 locus in an NF1+/− milieu composed by many other NF1 haploinsufficient (HI) cells, including mast cells, macrophages and myofibroblasts [1]. A distinctive feature of the Pnf is a rigid extracellular matrix (ECM) structure, as myofibroblasts stimulated by TGF-β-secreting mast cells produce high quantities of insoluble proteins such as fibronectin and different types of collagens [3,4]. This causes both SC proliferation and deposition of an abnormal ECM. Among MEK inhibitors, Selumetinib has been recently approved for Pnf treatment by the Food and Drug

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